The long-term goal of this proposal is to elucidate the molecular structure of the exocytotic fusion pore, the water-filled connection that forms between the lumen of a secretory granule and the extracellular space, upon exocytosis. Understanding the structure and regulation of the exocytotic fusion pore is one of the most outstanding questions on the mechanisms of exocytotic secretion. Towards this aim we will measure the time course of the conductance of single fusion pores. Pore conductance will be measured from the changes in the admittance of single patch-clamped mast cells caused by the formation of individual fusion pores as the cell undergoes exocytosis. We will study exocytotic fusion pores that form transiently and fusion pores that, after formation, expand irreversibly, completing exocytosis. We will also study the pores that form in the final stages of membrane retrieval events. We will classify and characterize the stages of development and the properties of the three types of pores. We will attempt to perturb each stage gain knowledge about the participating molecular structures. We will perturb pore structure by changing the physical properties of the mast cell membrane through changes in temperature, addition of anesthetics or fatty acid supplements; by chemical regulators like GTPgammaS and Ca++; and by genetic mutations like those of the pigment mutant Ruby. The result of these manipulations will be the basis for proposing models of the structure of the fusion pore as it evolves to complete the exocytotic event.